This invention relates to a process for preparing dipeptides having a free carboxyl group. More specifically, this invention relates to a process for preparing the dipeptides by contacting amino acids in the presence of a protease and a polyol.
The enzyme catalyzed formation of peptide bonds using a protease is well known. Since this is a reversible reaction, various methods have been used to favor the formation of the peptide bond. These include the use of organic cosolvents as reported by Homandberg et al. ("Synthesis of Peptide Bonds by Proteinases. Addition of Organic Cosolvents Shifts Peptide Bond Equilibria Toward Synthesis", Biochemistry, Vol. 17, No. 24, p 5220, 1978). Homandberg et al. reported that cosolvents such as 1,4-butanediol, ethylene glycol, and triethylene glycol shifted the equilibrium to favor peptide bond formation.
The formation of peptides using amino acids having an N-terminal protective group and amino acids having a C-terminal ester group with enzymes such as papain and prolisin has been reported by Isowa et al. in the Bulletin Of the Chemical Society of Japan, Vol. 50 (10), 2766-2772 (1977). Isowa et al. also disclosed the thermolysin catalyzed reactions of N-protected but side chain unprotected acidic amino acids with phenylalanine alkyl esters.
U.S. Pat. No. 4,165,311 of Isowa et al. (1979) discloses a process for preparing a salt (hereinafter referred to as the "adduct") having the formula: ##STR1## Wherein R.sub.1, R.sub.2, and n are as described hereinbelow and R.sub.3 is an alkoxy, benzyloxy, or benzhydryloxy group. This patent discloses that it is necessary to form the water insoluble adduct in order to drive the reversible reaction toward the peptide formation.
U.S. Pat. No. 4,256,836 of Isowa et al. (1981) discloses that it is necessary to treat the adduct with a strong acid solution, such as hydrochloric acid to free the dipeptide product from the amino acid ester. The dipeptide product is then extracted into an organic solvent and recovered. The process is disclosed as being useful for preparing LL-aspartylphenylalanine methyl ester, the synthetic sweetener.
The process disclosed in these two patents has the disadvantage of requiring additional steps to decompose the adduct in order to obtain the dipeptide product. Also, a significant excess of the esterified amino acid component (on the order of at least 2:1) is required in order to form the adduct with the dipeptide product. This excess must then be recycled back to the process in order for the process to be economical. Accordingly, there is a need for a process which eliminates the formation of this adduct and allows the preparation and recovery of the dipeptide product directly.